The present invention relates to novel modified tissue inhibitors of metalloproteinase (hereinafter referred to as xe2x80x9cTIMPsxe2x80x9d).
The subject application claims priority based on the Japanese Patent Application No. 095142/1999. The contents of the Japanese application is hereby incorporated by reference.
Metastasis is a feature of malignant cancer and most life-threatening pathologies and therefore one of the important objects of cancer therapy is to arrest metastasis. In practice, metastasis is palliatively treated by surgery, radiation therapy or chemotherapy, but no therapy can definitively arrest it. However, the mechanism of metastasis has been gradually elucidated in recent years and the breakdown system of extracellular matrix (hereinafter referred to as xe2x80x9cECMxe2x80x9d) is noted as a reflection of the metastatic potency of cancer.
More specifically, cancer cells begin to grow at the primary location and some of them discontinue adhering to surrounding cells so that they can escape from tumor tissues. However, tumor tissues are surrounded by dense ECM, and cancer cells cannot escape from there only via attack by physical motion without enzymatic breakdown. Metastatic cancer cells begin to move in the tissues by producing an enzyme that breaks down this barrier ECM. To further move to a distant location, cancer cells break vascular walls formed by robust ECM to enter the bloodstream. Then, they adhere to the inner membranes of the vascular walls at the second location, and enzymatically break down the vascular wall ECM again to escape from the vessel and infiltrate tissues by further breaking down surrounding ECM (xe2x80x9cSAIBOU KOUGAKUxe2x80x9d (Cell Technology), Vol. 17, No. 4, 1998, pp. 523-533).
In such a cascade of processes, breakdown of EMC seems to be most important for studying or diagnosing metastasis of cancer cells. Matrix metalloproteinases (hereinafter referred to as xe2x80x9cMMPsxe2x80x9d) (Docherty, A. J. P., O""Connell, J., Crabbe, T., Angal, S. and Murphy, G. (1992) Trends Biotechnol. 10, 200-207) are zinc-dependent endopeptidases that degrade components of ECM. MMPs play an essential role in tissue remodeling under physiological and pathological conditions such as morphogenesis, angiogenesis, tissue repair and tumor invasion (Docherty, A. J. P. et al., (1992) Trends Biotechnolol. 10, 200-207, supra.; Matrisian, L. M. (1992) Bioessays 14, 455-463; Stetler-Stevenson, W. G., Aznavoorian, S., and Liotta, L. A. (1993) Annu. Rev. Cell Biol. 9, 541-573). Most MMPs are secreted as zymogens and are activated by serine proteases or some activated MMPs.
At present, about twenty MMPs have been discovered, which have characteristic substrate specificities to degrade various collagens, glycoproteins, proteoglycans, etc. MMPs are grouped into several families by their substrate specificities and morphologies. For example, MMP-2 and -9 are also referred to as gelatinase A and gelatinase B, respectively, as members of the gelatinase family having a gelatin as a substrate. MMP-14 to -17 are the membrane-associated type, and belong to the MT-MMP family (membrane type-MMP). MMP-14 to -17 are referred to as MT1-MMP, MT2-MMP, MT3-MMP and MT4-MMP, respectively. Other families are the collagenase family (MMP-1, MMP-8, MMP-13 and MMP-18), stromelysin family (MMP-3 and MMP-10), etc.
The activities of activated MMPs are regulated by a family of specific inhibitors known as tissue inhibitors of metalloproteinases (hereinafter referred to as xe2x80x9cTIMPsxe2x80x9d). At present, four TIMPs have been identified, which efficiently inhibit MMPs except for MT-MMP. MT-MMP has selectivity in that it is efficiently inhibited by TIMP-2 and TIMP-3, but hardly inhibited by TIMP-1. TIMPs have a structure basically consisting of an N-terminal region and a C-terminal region. The MMP-inhibitory activity exists at the N-terminal region of TIMPs, and even recombinant TIMPs lacking the C-terminal region can inhibit MMPs.
Previous studies proposed hypotheses about the mechanism of MMP-inhibitory activity of TIMPs. For example, the following findings have been obtained about TIMP-2 and TIMP-1.
Among the MMP family, gelatinase A (MMP-2) and gelatinase B (MMP-9) are critical in the invasion of tumor cells across basement membranes because of their strong activity against type IV collagen, a major component of basement membranes (Liotta, L. A. (1986) Cancer Res. 46, 1-7; Collier. I. E., Wilhelm, S. M., Elsen, A. Z., Marmer, B. L., Grant G. A., Seltzer, J. L., Kronberger, A., He, C., Bauer E. A., and Goldberg, G. I. (1988) J. Biol. Chem. 263, 6579-6587; Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant G. A., and Goldberg, G. I. (1989) J. Biol. Chem. 264, 17213-17221). Unlike other zymogens of MMPs, progelatinase A is not activated by serine proteases or soluble MMPs and had been reported to be activated by a MMP-like activity on the surface of cancer and fibroblastic cells (Overall, C. M., and Sodek, J. (1990) J. Biol. Chem. 265, 21141-21151; Brown, P. D., Levy, A. T., Margulies, I. M., Liotta, L. A., and Stetler-Stevenson, W. G. (1990) Cancer Res. 50, 6184-6191; Ward, R V., Atkinson, S. J., Slocombe, P. M., Docherty, A. J., Reynolds, J. J., and Murphy, G. (1991) Biochim. Biophys. Acta. 1079, 242-246; Azzam, H. S. and Thompson, E. W. (1992) Cancer Res 52, 4540-4544).
Sato et al. (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65) identified a novel membrane-type MMP, named MT-MMP as an activator of progelatinase A on the cell surface. The cell-mediated activation of progelatinase A includes two steps of processing; MT-MMP-catalyzed cleavage of progelatinase A at a peptide bond between Asn-37 and Leu-38 firstly converts the zymogen into an intermediate form, and then autocatalytic cleavage of an Asn-80-Tyr-81 bond converts the intermediate form into a mature one (Strongin, A. Y., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1993) J. Biol. Chem. 268, 14033-14039). Several studies suggest that both steps are greatly accelerated by binding of (pro)gelatinase A onto the cell surface, and therefore, the receptor of (pro)gelatinase A on the cell surface is important for the activation. Carboxy-terminal hemopexin-like domain of gelatinase A is reported to be essential for the interaction with the cell-surface receptor (Strongin, A. Y., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1993) J. Biol. Chem. 268, 14033-14039; Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grant, G. A., and Goldberg, G. I. (1995) J. Biol. Chem. 270, 5331-5338).
Recent studies (Brooks, P. C., Silletti, S., von Schalscha, T. L., Friedlander, M., and Cheresh, D. A. (1996) Cell 92, 391-400; Kinoshita, T., Sato, H., Takino, T., Itoh, M., Akizawa, T., and Seiki, M. (1996) Cancer Res. 56, 2535-2538; Pei, D. Q., and Weiss, S. J. (1996) J. Biol. Chem. 271, 9135-9140; Will, H., Atkinson, S. J., Butler, G. S., Smith, B., and Murphy, G. (1996) J. Biol. Chem. 271, 17119-17213; Lichte, A., Kolkenbrock, H., and Tschesche, H. (1996) FEBS Lett. 397, 277-282) suggest that transmembrane domainless variants of MT-MMP convert progelatinase A to the intermediate form but hardly to the mature one. It is also reported that cell-mediated processing of mutant progelatinase A of which active site residue is replaced by site-directed mutagenesis, does not produce the mature form of the mutant (Atkinson, S. J., Crabbe, T., Cowell, S., Ward, R. V., Butler, M. J., Sato, H., Seiki, M., Reynolds, J. J., and Murphy, G. (1995) J. Biol. Chem. 270, 30479-30485; Sato, H., Takino, T., Kinoshita, T., Imal, K., Okada, Y., Stetler-Stevenson, W. G., and Seiki, M. (1996) FEBB Lett. 385, 238-240).
These studies suggest the importance of cell-associated activity of gelatinase A for the conversion of intermediate form of gelatinase A to its mature form.
On the other hand, the crystal structures of complexes of TIMPs and MMPs have also been studied.
The crystal structure of the complexformed between TIMP-1 and stromelysin suggests that the free xcex1-amino group and carbonyl oxygen of NH2-terminal Cys-1 of TIMP-1 coordinate the catalytic zinc atom of stromelysin, thus being involved in the inhibitory action (Gomis-Ruth, F. X., Maskos, K., Betz, M., Bergner, A., Huber, R., Suzuki, K., Yoshida, N., Nagase, H., Brew, K., Bourenkov, G. P., Bartunik, H., and Bode, W. (1997) Nature,389, 77-81). Quite recently, the crystal structure of the complex formed between TIMP-2 and catalytic domain of MT1-MMP was also determined (Fernandez-Catalan, C., Bode, W., Huber, R., Turk, D., Calvete, J. J., Lichte, A., Tschesche, H., and Maskos, K. (1998) EMBO J. 17, 5238-5248). However, the crystal structures of the two MMP-TIMP complexes also indicate that TIMPs have wide range contacts with the corresponding MMPs.
Previously, it had been reported that chemical modification of TIMP-1 with diethyl pyrocarbonate abolishes the inhibitory activity. The modified residues are His-95, His-144 and His-164 of TIMP-1, and the modification of His-95 has been proposed to be responsible for the loss of activity (Williamson, R. A., Smith. B. J., Angal, S., and Freedman, R. B. (1993) Biochim. Biophys. Acta. 1203, 147-154). However, a study based on site-directed mutagenesis has revealed that replacement of His-95 to glutamine does not affect the inhibitory activity of TIMP-1 (Williamson, R. A., Smith, B. J., Angal, S., and Freedman, R. B. (1993) Biochem. Biophys. Acta. 1203, 147-154). Furthermore, the H95Q mutant is still sensitive to the diethyl pyrocarbonate-treatment. So far, there is no explanation for the effect of diethyl pyrocarbonate on the TIMP-1 activity.
Thus, the mechanism of MMP-inhibitory activity of TIMPs has been explained by the formation of complexes between TIMPs and MMPs, and the N-terminal region of TIMPs seemed to be involved in the formation of such complexes. However, the details are unknown and any methods for effectively arresting metastasis of cancer have not been obtained from the explanation of such a mechanism.
An object of the present invention is to provide a novel modified TIMP. The NH2-terminal xcex1-amino group of the present TIMP is modified with an electron-accepting group to substantially lose the ability to bind to a metalloproteinase.
The modified TIMP of the present invention is preferably a modified TIMP-2.
In the present-invention, said electron-accepting group is preferably a carbamyl group.
The present invention also provides a method of inhibiting the formation of a complex including a TIMP by adding said modified TIMP. The method may be any of in vivo, in vitro or ex vivo method.
In the inhibiting method of the present invention, said-modified TIMP is preferably a modified TIMP-2 and the complex is one including MT-MMP, TIMP-2 and gelatinase A.
Another object of the present invention is to provide a pharmaceutical composition comprising said modified TIMP in combination with a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention is used for the inhibition of metastasis of cancer or the inhibition of vascularization.